Facilitation of the closure of large skin wounds (i.e. burns, traumatic injury, congenital reconstruction) by a variety of methods requires extensive expense and time. Methods currently in use include, but are not limited to, split-thickness grafts from the same individual, the use of specially treated cadaver skin, and autologous cultured skin equivalents. Due to the availability of these materials, it has become clinically feasible to treat skin wounds, particularly burn wounds, with cells cultured from the same patient. This method of treatment is both expensive and time consuming.
Although autologous grafting solves certain problems inherent in tissue transplantation, such as histocompatibility and the potential need for immunosuppressive agents, major problems still exist. For example, patients must not only suffer the harvesting of significant amounts of skin for autologous culturing, which in itself causes wounding, but must also wait up to six weeks before grafting back to the wound. For the patient, this is just the start of a long and trying process leading towards the healing of the wound.
The ability to treat skin wounds or congenital defects in which a significant amount of epithelial tissue has been lost or rendered nonfunctional remains an important issue among clinicians. With the advent of effective antibiotics, one major hurdle to effective wound healing, i.e. healing without infection, was effectively overcome. Thus, the use of antibiotics allowed for the routine use of surgical grafting techniques to be developed and applied to a wide range of wounds and defects. Recent history has seen the use of a variety of epithelial graft techniques which have contributed significantly to reducing the morbidity and mortality of individuals with severe skin wounds, including improved aesthetic results.
Clinical Aspects of Epithelial Grafts. Epithelial grafts fall into three main categories: allografts (same species), xenografts (different species), and autografts (same animal). Over the centuries xenografts from a variety of animals and birds have been used with wide ranging results. For the most part, little success was achieved with the use of different xenografts. The need for a viable alternative prompted the search for better graft material. Cadaver allografts are still in use today, but they are usually restricted to patients with extreme burns.
When available or practical, autografting from a healthy site on the individual to the wound site is the presently preferred treatment. This, however, may have some drawbacks. For instance, this leaves a donor site which must also be treated as a wound and can lead to increased morbidity of both the donor and graft sites. When only small amounts of tissue are used, free grafts may be transferred to sites that have an adequate blood supply and an intact and functional connective tissue base. For larger wound sites, pedicle grafts may be used. Pedicle grafts are initially allowed to remain attached to the donor site until an adequate collateral blood supply is developed before the connection to the donor site is excised.
For all of the previously described grafts, the success of the tissue graft is primarily dependent on: (a) immunological response to the graft; (b) size of the graft; (c) anatomical area of graft; (d) condition of underlying tissue at graft site; (e) condition of surrounding tissue at graft site; (f) thickness of graft; and (g) maintenance of sterility of graft tissue and graft site.
Cultured Epithelial Autografts. With advances in cell culture techniques came new ideas for tissue grafting. These advances in cell culture techniques have made it possible to culture keratinocytes (skin cells) taken from a biopsy of a patient and to ultimately transfer the resulting autologous cultured cells back to the same individual.
By using autologous cultured grafts, problems in organ transplantation procedures may be solved such as: (a) obtaining histocompatibility of matched tissues; and (b) lack of a graft donor site. In principle, this approach addresses many major problems of tissue transplantation. First, using the patient as their own source of transplant tissue, coupled with expansion of his/her cells in tissue culture, eliminates the problem of tissue availability in the majority of the patients who would benefit. Second, because a patient is treated with his/her own cells, an immunosuppressive mediator is not required, nor is there a requirement for a large donor site. This has opened up a new era in tissue transplantation, especially in the use of autologous cultured tissue grafts to treat severely burned patients. Yet, there are still some disadvantages, for example, the time and expense involved in culturing cells as well as the lack of available donor graft sites (e.g., burn patients with more than 60% tissue involvement). A need is recognized for a readily available source of graftable tissue which may be utilized in major trauma or burn cases.
Discussion of Epithelial Graft Construction. While the use of cultured cells in treatment of burns patients is now a routine clinical procedure, several problems remain to be solved. When epithelial cells are harvested from biopsy material, the cells that proliferate in culture are mainly connective tissue fibroblasts and keratinocytes. Sweat glands, sebaceous glands, pigment cells, and other cell types that are usually required for a fully functional skin are lost during cell cultivation and, as a result, cell culture derived autologous skin may lack several physiologically important properties.
The development of tissue engineered epithelial grafts for use in wound repair is an aggressively researched area. While the use of cultured cells in the treatment of burn patients is now an accepted clinical procedure several problems still remain to be solved. The time between the formation of the wound and the application of the graft material has a significant effect on scar formation and re-epithelialization. Using early culture techniques for the stratification of keratinocytes in vitro in the production of skin grafts, the stratification was limited to only a few cell layers without keratinization. Later techniques allowed further differentiation of the keratinocytes into a thicker stratified layer. Recently, keratinization of the cultured epithelial tissue was accomplished by growing confluent stratified cultures at the gas/liquid interface of the culture medium.
Because these tissues had minimum shear strength due to thickness (<0.5 mm), the grafting of such tissue required the use of a pressure bandage to hold the graft in place until a basement membrane had formed which attached the graft to the wound surface. These grafts were also limited by the type of wound, in that they were only useful as analogues of split-thickness autografts. Split-thickness grafts differ from full-thickness grafts in that the former contains little, if any, tissue below the basement membrane on which the epithelium attaches to the dermis. Therefore split-thickness autografts require grafting sites containing a healthy connective tissue layer. To date no such graft has ever formed secondary structural morphology such as rete ridges or appendageal structures. The lack of such structures makes the grafts highly sensitive to trauma and infection.
The reconstruction of full-thickness grafts from cultured cells has had limited success and, up until recently, only when autologous donor collagen was used. Recent reports using dermal allografts have had some success, such as the grafting of full-thickness cultured oral mucosal cells in the mouse and dog. This was accomplished by the construction of a bilayer graft containing autologous cultured keratinocytes grown directly on a collagen-gel interspersed with autologous cultured fibroblasts.
The two major drawbacks to this method have been: (a) the lack of secondary structural morphology (rete ridge formation); and (b) the latent shrinking of the grafted collagen layer. The latter complication has been the most difficult problem in the clinical use of autologous cultured synthetic grafts resulting in the occasional loss of the graft. Researchers have tried to overcome this hurdle by combining the advances in graft tissue design techniques with the use of a cross-linked collagen-GAG matrix and the use of dermal allografts. Preliminary reports using this technique have shown rudimentary rete ridge formation and a decrease in graft contracture.
Methods to combine the technology of tissue engineering with that of dermal allografts have as yet not been developed. Studies attempting to combine synthetic full-thickness grafts with that of biodegradable polymers and copolymers are in the early stages of development. However, there are many studies utilizing a variety of natural and synthetic materials for use as a matrix support substratum for tissue reconstruction and augmentation. The field of tissue engineering is rapidly gaining ground as an alternative to aggressive surgical techniques for the repair of wounds and other deformities.